Cartilage has limited intrinsic healing potential upon injury, due to the low cell density and the lack of blood supply. Degenerative disease of the cartilage, such as osteoarthritis (OA), is challenging to treat without clear mechanistic understandings of cartilage development. With over 90% of the cartilage tissue occupied by extracellular matrix (ECM), understanding the cellular and molecular effects of cartilage ECM on chondrogenesis and chondrocyte behavior is crucial for therapeutic development. The focus of this work is to study the regulation of chondrogenesis and hypertrophic maturation of human mesenchymal stem cells (MSCs) by cartilage ECM in the context of potential therapeutic applications. To study the cartilage ECM, we created a decellularized ECM digest from native porcine cartilage and examined its effects on MSCs. Since native cartilage ECM maintains chondrocyte homeostasis without progressing to hypertrophic degeneration, we hypothesized that the decellularized ECM would promote MSC chondrogenesis and inhibit hypertrophy. Indeed, we showed that ECM promoted MSC chondrogenesis and matrix production, and inhibited hypertrophy and endochondral ossification. The chondrogenic effect was shown to potentially involve the PI3K-Akt-Foxo1 and Hif1 pathways. By recapitulating the activated Hif1 pathway, roxadustat, a small molecule stabilizer of Hif, was able to reproduce the chondrogenic and anti-hypertrophic effects of the cartilage ECM. It also reduced the expression of matrix metalloproteases (MMPs) in MSCs, healthy chondrocytes, and OA chondrocytes, and alleviated matrix degradation in bovine cartilage explants. We also attempted to identify ECM components that display chondrogenic properties. Collagen XI, a minor component of articular cartilage, was shown to promote cartilage matrix formation in MSCs and healthy chondrocytes, and to reduce matrix degradation in bovine cartilage explants. Taken together, this study reveals the dual roles of cartilage ECM in promoting chondrogenesis and matrix production and inhibiting cartilage hypertrophy. Importantly, small molecule drugs that recapitulate the signaling pathways of ECM regulation, and collagen XI, a component of the ECM, may serve as leads for further therapeutic development for cartilage injury and degenerative disease.

by Chi-Kwan Leung. / "July 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 106-109). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

In periodontitis, there is a disruption in the homeostasis of the oral microbiome by peridontopathogenic bacteria. However, while bacteria is essential for periodontitis to occur, the severity, pattern and progression of the disease is not solely determined by the microbial burden, and in fact has a lot to do with the overwhelming host inflammatory response. The response can vary even in two individuals with similar periodontopathogenic profiles. The host response leads to extracellular matrix (ECM) destruction, loss of attachment, alveolar bone resorption and eventually, edentulism. The host's reaction is orchestrated by proinflammatory cytokines and chemokines and matrix metalloproteinases (MMPs). MMPs are proteolytic enzymes capable of degrading collagen fibers from the extracellular matrix and are the main responsible for tissue damage and gingival recession in periodontitis. As a response to the limitations of the traditional therapies, new agents have been used in preclinical and clinical studies, namely host-modulatory agents, including anti-proteinase agents, anti-inflammatory agents and anti-resorptive agents. Focusing on changing the inflammatory process, as opposed to the microbial insult, can slow down the disease progression, improve clinical outcomes and even prevent tooth loss in severely compromised patients. This work examines the role of pro-inflammatory markers homocysteine in chronic inflammation and periodontitis. Homocysteine (Hcy) is a non-protein amino acid derived from the metabolism of the essential amino acid methionine via methyl group metabolism. Controlling Homocysteine as a potential inductor of MMPs, and hence of tissue destruction, can lead to new adjuvant therapies to improve clinical outcomes and prevent activation of the disease

We define the Lie group G2 and show several equivalent ways to view G2. We do the same with its Lie algebra g2. We identify a new basis for g2 using Bryant’s view of g2 and geometric considerations we develop. We then show how to construct a matrix representation of G2 given our particular basis for g2. We examine the geometry of 1 and 2-parameter subgroups of G2. Finally, we suggest an area of further research using the new geometric characterization we developed for g2.

The infectious first-stage larvae of the nematode Trichinella spiralis is an intracellular parasite of altered skeletal muscle. Invasion of the muscle cell initiates a series of morphological changes in the host muscle cell which ultimately results in a specialized unit called the nurse cell. The completed nurse cell consists of a collagenous capsule, matrix of altered sarcoplasm, and a circulatory rete. The purpose of this study was to determine the types of collagen present in the nurse cell capsule. Additionally, the presence of the gl ycoproteins, laminin and tenascin was determined. This study also sought to demonstrate the location of the selected extracellular matrix proteins within the capsule. Nurse cells were isolated from infected host muscle by sequential protease treatment with pronase, collagenase, and hyaluronidase. Nurse cells were digested with pepsin to produce characteristic pepsin-resistant triple helical fragments of collagen. The nurse cellpepsin digest was characterized by SDS-page, under reduced and nonreduced conditions, with type VI collagen and the ala2a3 chains of type XI collagen. Frozen tissue sections of infected and non-infected rat diaphragms were screened with specific polyclonal antibodies against types I, m, IV, V/Xl, and VI collagen, laminin, and tenascin. Indirect immunofluorescence using FITC secondary antibodies was used to locate the protein in the capsule and host tissue. SDS-page of the nurse cell-pepsin digest produced an electrophoretic pattern of resistant fragments characteristic for types I, III, IV, V, and VI collagen. Additionally, fragments migrated with an apparent molecular weight expected for pepsin resistant fragments of laminin. Indirect immunofluorescence showed types I, III, IV, and VI collagen, and laminin were distributed throughout the capsule. Serum No. 4876, which recognizes type V /XJ collagen, localized to the larvae. Tenascin failed to stain the nurse cell or host tissue. The results show that the capsule is a heterogenic structure with types I, III, IV, V, and VI collagens, and laminin distributed throughout the structure. The immunolocalization of Serum No. 4876 to the larvae suggests that a nematode collagen shares an amino acid sequence in common with mammalian type V /XI collagen.

Human adult cartilage is an aneural and avascular type of connective tissue, which consequently reflects reduced growth and repair rates. The main cell type of cartilage are chondrocytes, previously derived from human mesenchymal stem cells (hMSCs). They are responsible for the production and maintainance of the cartilaginous extracellular matrix (ECM), which consists mainly of collagen and proteoglycans. Signal transmission to or from chondrocytes, generally occurs via interaction with signalling factors connected to the cartilaginous ECM. In this context, proteins of the CCN family were identified as important matricellular and multifunctional regulators with high significance during skeletal development and fracture repair. In this thesis, main focus lies on WISP1/CCN4, which is known as a general survival factor in a variety of cell types and seems to be crucial during lineage progression of hMSCs into chondrocytes. We intend to counter the lack of knowledge about the general importance of WISP1-signalling within the musculoskeletal system and especially regarding cell death and survival by a variety of molecular and cell biology methods. First, we established a successful down-regulation of endogenous WISP1 transcripts within different cell types of the human musculoskeletal system through gene-silencing. Interestingly, WISP1 seems to be crucial to the survival of all examined cell lines and primary hMSCs, since a loss of WISP1 resulted in cell death. Bioinformatical analyses of subsequent performed microarrays (WISP1 down-regulated vs. control samples) confirmed this observation in primary hMSCs and the chondrocyte cell line Tc28a2. Distinct clusters of regulated genes, closely related to apoptosis induction, could be identified. In this context, TRAIL induced apoptosis as well as p53 mediated cell death seem to play a crucial role during the absence of WISP1 in hMSCs. By contrast, microarray analysis of WISP1 down-regulated chondrocytes indicated rather apoptosis induction via MAPK-signalling. Despite apoptosis relevant gene regulations, microarray analyses also identified clusters of differentially expressed genes of other important cellular activities, e.g. a huge cluster of interferon-inducible genes in hMSCs or gene regulations affecting cartilage homeostasis in chondrocytes. Results of this thesis emphasize the importance of regulatory mechanisms that influence cell survival of primary hMSCs and chondrocytes in the enforced absence of WISP1. Moreover, findings intensified the assumed importance for WISP1-signalling in cartilage homeostasis. Thus, this thesis generated an essential fundament for further examinations to investigate the role of WISP1-signalling in cartilage homeostasis and cell death. / Humaner adulter Knorpel besitzt weder Blutgefäße noch Nerven, weswegen diese Knorpelart im Vergleich zu anderen Gewebetypen ein verringertes Wachstum und Regenerierung wiederspiegelt. Den Hauptteil der Zellen im adulten Knorpel stellen die Chondrozyten (Knorpelzellen)dar, welche sich zuvor aus humanen mesenchymalen Stammzellen (hMSCs) entwickelt haben. Sie sind verantwortlich für die Bildung und Aufrechterhaltung der extrazellulären Matrix (ECM) des Knorpelgewebes, welche hauptsächlich aus Kollagen und Proteoglykanen besteht. Signale, die durch Chondrozyten erzeugt oder weitergeleitet werden, finden in der Regel durch Interaktion mit Molekülen der im Knorpel liegenden ECM statt. Mitglieder der CCN-Familie gelten hierbei als bedeutende extrazelluläre Matrixproteine, die bei verschiedenen regulatorischen Prozessen während der Skelettentwicklung und der Frakturheilung eine Rolle spielen. In dieser Doktorarbeit liegt das Hauptaugenmerk auf dem CCN Protein WISP1/CCN4. Dieses Protein gilt bereits in verschiedenen Zellen als ein notwendiger Überlebensfaktor und scheint desWeiteren eine regulatorische Funktion während der Differenzierung von hMSCs in Chondrozyten auszuüben. Die generelle Bedeutung von WISP1 für das muskuloskelettale System ist bislang jedoch weitgehend ungeklärt und soll während dieser Doktorarbeit mittels einer Reihe von molekular- und zellbiologischer Methoden genauer untersucht werden. Hierfür wurde zu Beginn eine erfolgreiche Herunterregulierung endogen hergestellter WISP1 Transkripte mittels Genexpressionshemmung (gene-silencing) in verschiedenen muskuloskelettalen Zellen erzielt. Interessanterweise scheint WISP1 eine bedeutende Rolle für das Überleben dieser Zellen zu spielen, da ein Verlust bei allen untersuchten Zelllinien und primären hMSCs zum Zelltod führte. Um zu Grunde liegende Mechanismen genauer zu untersuchen, wurden daraufhin Microarray Analysen von hMSCs und Tc28a2 Chondrocyten durchgeführt (jeweils WISP1 herunterreguliert vs Kontrollzellen). In diesem Zusammenhang identifizierten bioinformatische Analysen differentielle Expressionen verschiedener apoptoseresponsiver Gene. So scheint eine Apoptoseinduktion über TRAIL und/oder p53 in hMSCs stattzufinden, wohingegen eine starke Regulation des MAPK-Signalweges in Chondrozyten detektiert wurde. Neben diesen Genregulationen, deckten die Analysen ebenso Gengruppen auf, die bei anderen wichtigen zellulären Abläufen eine Rolle spielen. Hier sind in WISP1 herunterregulierten hMSCs u.a. viele differenziell exprimierte Gene zu nennen, die durch Interferone induzierbar sind. In Chondrozyten dagegen scheint eine verringerte WISP1 Expression Genexpressionen zu beeinflussen, welche die Knorpelhomeostase regulieren. Die Ergebnisse, die während dieser Doktorarbeit erzielt wurden, verdeutlichen die Wichtigkeit von WISP1 für das Überleben von primären hMSCs und Chondrozyten. Darüberhinaus verstärken die bioinformatischen Analysen die Annahme, das WISP1 regulatorische Funktionen für die Knorpelhomeostase ausübt. Somit bietet diese Doktorarbeit ein essentielles Fundament, um die Rolle von WISP1 bei der Aufrechterhaltung der Knorpelhomeostase und des Zelltodes weiter zu erforschen.

In this thesis, we focus on studying the performance of general regularized discriminant analysis (RDA) classifiers. The data used for analysis is assumed to follow Gaussian mixture model with different means and covariances. RDA offers a rich class of regularization options, covering as special cases the regularized linear discriminant analysis (RLDA) and the regularized quadratic discriminant analysis (RQDA) classi ers. We analyze RDA under the double asymptotic regime where the data dimension and the training size both increase in a proportional way. This double asymptotic regime allows for application of fundamental results from random matrix theory. Under the double asymptotic regime and some mild assumptions, we show that the asymptotic classification error converges to a deterministic quantity that only depends on the data statistical parameters and dimensions. This result not only implicates some mathematical relations between the misclassification error and the class statistics, but also can be leveraged to select the optimal parameters that minimize the classification error, thus yielding the optimal classifier. Validation results on the synthetic data show a good accuracy of our theoretical findings. We also construct a general consistent estimator to approximate the true classification error in consideration of the unknown previous statistics. We benchmark the performance of our proposed consistent estimator against classical estimator on synthetic data. The observations demonstrate that the general estimator outperforms others in terms of mean squared error (MSE).

No description available.

The expected yield to maturity on a defaultable obligation equals the nominal yield less expected default losses. However, in a mark-to-market world, one doesn't have the luxury of reporting one's performance on the basis of yield to maturity. Total return is calculated for an arbitrary holding period, and must reflect any mark-to-market gains or losses as at the close of the period-gains or losses that can be triggered by the bond's upgrade or downgrade. Thus to estimate expected total return, one must estimate not only expected default losses, but also the impact on capital price of expected ratings transitions. This paper begins with the observation that a bond which is blessed by more favourable transition characteristics is likely to produce a higher total return, and poses the question of how that benefit can be quantified. How much is it worth? To answer the question, I start by specifying a formal bond-pricing model reflective of ratings transitions. I survey various statistical methods and past research efforts to identify the ratings-transition matrix which best parametrises the model, and propose a novel test for selecting between competing matrices. Using this approach, I replicate several important studies of ratings transitions. I also use it to examine new published and unpublished data, testing for (and finding) ratings path-dependency, and otherwise exploring the effect of ratings changes on different bond sectors. I then turn to the question of whether it is possible to estimate bond-specific transition probabilities, and propose a way to do so. I combine these efforts into the specifications for a pricing model capable of answering the question: How much is it worth?

A detailed row by row investigation of major lateral tooth cusp mineralisation, together with the concomitant development of the superior epithelial tissue surrounding the teeth of the chiton Acanthopleura hirtosa has been undertaken using a combination of light microscopy, and scanning and transmission electron microscopy. A holistic approach has been adopted that encompasses observations over a range of spatial scales, from whole radula mineralisation processes to those occurring within individual tooth cusps at various stages of development. In addition, mineralisation in radulae from freshly collected animals has been compared to that of animals maintained for extensive periods within a newly developed iron limited system, which restricts radula mineralisation without impeding the formation of the organic matrix. An evaluation of the iron limitation technique has revealed that maintaining specimens of A. hirtosa within an iron poor environment results in a significant departure from the normal pattern of mineralisation in these animals. As a consequence of iron limitation, there is an obvious increase in the number of unmineralised tooth rows in addition to associated alterations in structure and composition at all stages of tooth development. In normal specimens of A. hirtosa, the onset of mineralisation in the tooth cusps occurs following the prior accumulation of iron at the junction zone and the sudden accumulation of iron containing granules in the cusp epithelium at tooth row 13. The superior epithelium surrounding the tooth cusps undergoes a series of developmental changes leading up to, and following, the onset of mineralisation. In particular, the abundance of mitochondria within the apical cusp epithelium increases, presumably in order to provide the ideal conditions of pH, and thus solubility, needed for the supersaturation of iron and its nucleation at row 13. Once mineralisation has commenced, the microvilli attached to the cusps develop rapidly, and are suggested to do so in order to facilitate the transport of iron, and thereby ensure that a high concentration gradient of this element into the cusps is maintained. The delivery of iron into the cusps occurs from two fronts, the first from the superior epithelium via the posterior surface, and the second from the junction zone via an internal pathway situated along the lepidocrocite boundary between the magnetite and core regions of the tooth. The existence of a plume of elements between this internal mineralisation pathway and the junction zone, provides the first direct evidence that the junction zone is involved in the storage and release of elements for cusp mineralisation. Data from iron limited radulae also indicate that iron continues to be deposited at the junction zone in preference to the superior epithelium or cusps, despite the disruption of mineralisation, highlighting the importance of this region in the mineralisation process. Iron reinstatement experiments have also shown that the internal pathways of iron delivery within the organic matrix remain viable, despite prolonged periods of iron limitation. In addition, the reinstatement of iron has revealed that the plumes, situated between the junction zone and internal mineralising pathway of the cusp, stem from the centre of the plate like junction zone, directly above the stylus canal, a tube like cavity situated within the styli of each major lateral tooth. An in depth study of the stylus canal has revealed that cells within the canal are remarkably similar to those of the epithelium surrounding the cusps, suggesting that this structure may also be involved in the delivery of ions to the junction zone. The stylus canal is shown to be present in the major lateral tooth cusps of 38 chiton species distributed worldwide, and is therefore likely to be a feature common to all chitons. The presence of the canal, and indeed its absence from the bases of all remaining non iron mineralised teeth, irrespective of chiton species, also points strongly to a functional relationship between the stylus canal and tooth cusp mineralisation.